Possible antimicrobial effects of all samples were eliminated and validated following the method proposed by the USP under validation of microbial recovery from pharmacopeial articles using Fluid Casein Digest-Soy LecithinPolysorbate 20 medium (Merck) (14, 15). The unused eye-drops were subjected to the BP (10) and USP (11) preservative challenge tests using Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404 as test organisms. To determine the microbial killing rate, the eyedrops were inoculated with challenging microorganisms at a final concentration of 105 -106 CFU ml-1 and the viable organisms were determined 30, 90 and 180 min after inoculation for bacteria and 24 hrs for fungi.Aerobic viable cell count of 1:10 dilution of product in neutralizer was determined by plate count method and 0.5 log increase in colony forming units was accounted for variability.